Regulation at the level of RNA governs organismal phenotype 1-3. RNA binding proteins (RBPs) are key actors in this regulation, and RBPs of the Puf family are exemplars of post-transcritpional gene regulation 4,5. One way Pufs act is to bind cytoplasmic mRNAs in a sequence-specific manner, thereby recruiting the conserved Pop2- Ccr4 deadenylase complex and causing translational repression 7,8. However, Puf proteins are also known to possess Pop2-Ccr4 deadenylation-independent activity, though this alternate mechanism of action is largely undescribed 7-12, this work. Further, how and why a yeast Puf protein adopts alternate mechanisms of action, and whether a target RNA is subject to only one mode of regulation or to multiple modes depending on context remains uninvestigated. The goal of this proposal is to characterize alternate modes of Puf-mediated protein repression. We examine three Pumilio-like Puf proteins in the model yeast S. cerevisiae, Puf3p Puf4, and Puf5: a) Identify protein binding partners necessary for Puf-mediated translational repression of target mRNAs. We will couple systematic mutant analysis with forward genetic screens to find new Puf protein interactors, creating targeted mutations in conserved amino acids to identify translation-defective Puf mutants not compromised in target RNA binding, and will then rescue these mutants with high copy suppressor screens. b) Characterize differences between deadenylation-dependent and -independent Puf translational repression by identifying substrates associated with each mechanism. We propose a novel method (poly-U polymerase tagging) to identify both total RNA Puf substrates, and via anti-Ccr4 immunoprecipitation, substrates bound specifically by the Ccr4 deadenylase. We hypothesize that only a subset of RNAs bound by Puf are also regulated by Ccr4p, and their identity helps illuminate the functional importance of each mechanism. c) Investigate how Puf protein-mediated translational repression is initially established and determine whether Puf3p, Puf4p, and Puf5p affect RNA fate in the nucleus. Regulation of gene expression occurs both in the nucleus and the cytoplasm of cells. We hypothesize that cytoplasmic Puf proteins initiate translational control in the nucleus. We will determine whether Puf3p, Puf4p, or Puf5p are localized to yeast nuclei, perform chromatin immunoprecipitation to establish whether and at what genomic locations Pufs may be associated with, determine whether these Puf proteins are associated with RNA in the nucleus, and investigate whether they are exported through nuclear pores out into the cytoplasm bound to target RNAs via microscopy. Puf proteins are found throughout eukaryotes, and regulation of RNA is common to eukaryotes as well. Thus, the Puf protein interactors and mechanisms of action we uncover in S. cerevisiae may also be widespread. As many human diseases are caused by defects in RNA binding proteins (e.g., fragile X syndrome, muscular dystrophy, and sterility ) ,understanding general mechanisms through which RNAs are controlled is likely to shed light on the etiology of such diseases and on the ways in which RBP defects could be circumvented.